Modified arginine deiminase

ABSTRACT

The present invention is directed to arginine deiminase modified with polyethylene glycol, to methods of treating cancer, and to methods of treating and/or inhibiting metastasis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a related to U.S. patent application Ser. No.09/023,809, allowed, which claims priority to U.S. ProvisionalApplication Ser. No. 60/046,200, filed on May 12, 1997, each of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to arginine deiminase modified withpolyethylene glycol, to methods for treating cancer, and to methods fortreating and/or inhibiting metastasis.

BACKGROUND OF THE INVENTION

Malignant melanoma (stage 3) and hepatoma are fatal diseases which killmost patients within one year of diagnosis. In the United States,approximately 16,000 people die from these diseases annually. Theincidence of melanoma is rapidly increasing in the United States and iseven higher in other countries, such as Australia. The incidence ofhepatoma, in parts of the world where hepatitis is endemic, is evengreater. For example, hepatoma is one of the leading forms of cancer inJapan and Taiwan. Effective treatments for these diseases are urgentlyneeded.

Selective deprivation of essential amino acids has been used to treatsome forms of cancer. The best known example is the use ofL-asparaginase to lower levels of asparagine as a treatment for acutelymphoblastic leukemia. The L-asparaginase most frequently used isisolated from E. coli. However, clinical use of this enzyme iscompromised by its inherent antigenicity and short circulatinghalf-life, as described by Y. K. Park, et al, Anticancer Res., 1:373-376(1981). Covalent modification of E. coli L-asparaginase withpolyethylene glycol reduces its antigenicity and prolongs itscirculating half-life, as described, for example, by Park, AnticancerRes., supra; Y. Kamisaki et al, J. Pharmacol. Exp. Ther., 216:410-414(1981); and Y. Kamisaki et al, Gann., 73:47-474 (1982). Although therehas been a great deal of effort to identify other essential amino aciddegrading enzymes for the treatment of cancer, none have been approved,primarily because deprivation of essential amino acids, by definition,results in numerous, and severe, side effects.

It has been reported that enzymes which degrade non-essential aminoacids, such as arginine, may be an effective means of controlling someforms of cancer. For example, arginine deiminase (ADI) isolated fromPseudomonas pudita was described by J. B. Jones, “The Effect of ArginineDeiminase on Murine Leukemic Lymphoblasts,” Ph.D. Dissertation, TheUniversity of Oklahoma, pages 1-165 (1981). Although effective inkilling tumor cells in vitro, ADI isolated from P. pudita failed toexhibit efficacy in vivo because it had little enzyme activity at aneutral pH and was rapidly cleared from the circulation of experimentalanimals. Arginine deiminase derived from Mycoplasma arginini isdescribed, for example, by Takaku et al, Int. J. Cancer, 51:244-249(1992), and U.S. Pat. No. 5,474,928, the disclosures of which are herebyincorporated by reference herein in their entirety. However, a problemassociated with the therapeutic use of such a heterologous protein isits antigenicity. The chemical modification of arginine deiminase fromMycoplasma arginini, via a cyanuric chloride linking group, withpolyethylene glycol was described by Takaku et al., Jpn. J. Cancer Res.,84:1195-1200 (1993). However, the modified protein was toxic whenmetabolized due to the release of cyanide from the cyanuric chloridelinking group.

There is a need for compositions which degrade non-essential amino acidsand which do not have the problems associated with the prior art. Thepresent invention is directed to these, as well as other, importantends.

SUMMARY OF THE INVENTION

The present invention is directed to arginine deiminase modified withpolyethylene glycol. In a preferred embodiment, the arginine deiminaseis modified with polyethylene glycol, having a total weight averagemolecular weight of about 1,000 to about 50,000, directly or through abiocompatible linking group.

Another embodiment of the invention is directed to methods of treatingcancer, including, for example, sarcomas, hepatomas and melanomas. Theinvention is also directed to methods of treating and/or inhibiting themetastasis of tumor cells.

These and other aspects of the present invention will be elucidated inthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of arginine deiminase clonedfrom Mycoplasma arginini (the top amino acid sequence SEQ ID NO: 1,identified as ADIPROT), Mycoplasma arthritides (the middle amino acidsequence SEQ ID NO: 2, identified as ARTADIPRO), and Mycoplasma hominus(the bottom amino acid sequence SEQ ID NO: 3, identified as HOMADIPRO).

FIGS. 2A and 2B are graphs showing the effect of a single dose of nativearginine deiminase and arginine deiminase modified with polyethyleneglycol (e.g., molecular weight 5,000) on serum arginine levels and serumcitrulline levels in mice.

FIG. 3 is a graph showing the effects on serum arginine levels whenPEG10,000 is covalently bonded to ADI via various linking groups.

FIG. 4 is a graph showing the effect that the linking group and themolecular weight of the polyethylene glycol have on citrullineproduction in mice injected with a single dose of PEG-ADI.

FIGS. 5A and 5B are graphs showing the dose response thatADI-SS-PEG5,000 had on serum arginine and citrulline levels.

FIGS. 5C and 5D are graphs showing the dose response thatADI-SS-PEG20,000 had on serum arginine and citrulline levels.

FIG. 6 is a graph showing the antigenicity of native ADI,ADI-SS-PEG5,000, and ADI-SS-PEG20,000.

FIG. 7 is a graph showing the effect that treatments withADI-SS-PEG5,000, ADI-SS-PEG12,000 or ADI-SS-PEG20,000 had on tumor sizein mice which were injected with SK-mel 2 human melanoma cells.

FIG. 8 is a graph showing the effect that treatments with ADI-PEG20,000had on tumor size in mice which were injected with SK-mel 28, SK-mel 2or M24-met human melanoma cells.

FIG. 9 is a graph showing the effect that treatments with ADI-PEG5,000,ADI-PEG12,000 or ADI-PEG20,000 had on the survival of mice which wereinjected with human hepatoma SK-Hep1 cells.

FIG. 10 depicts the amino acid sequences of arginine deiminase clonedfrom Steptococcus pyogenes (the top amino acid sequence SEQ ID NO: 6,identified as STRADIPYR) and Steptococcus pneumoniae(the bottom aminoacid sequence SEQ ID NO: 7, identified as STRADIPNE).

FIG. 11 depicts the amino acid sequences of arginine deiminase clonedfrom Borrelia burgdorferi (the top amino acid sequence SEQ ID NO: 8,identified as BORADIBUR) and Borrelia afzelii (the bottom amino acidsequence SEQ ID NO: 9, identified as BORADIAFZ).

FIG. 12 depicts the amino acid sequence of Qiardia intestinalis (the topamino acid sequence SEQ ID NO: 10, identified as QIAADIINT), Clostridiumperfringens (the middle amino acid sequence SEQ ID NO: 11, identified asCLOADIPER) and Bacillus licheniformis (the bottom amino acid sequenceSEQ ID NO: 12, identified as BACADILIC).

FIG. 13 depicts the amino acid sequence of Enterococcus faecalis (thetop amino acid sequence SEQ ID NO: 13, identified as ENTADIFAE) andLactobacillus sake (the bottom amino acid sequence SEQ ID NO: 14,identified as LACADISAK).

DETAILED DESCRIPTION OF THE INVENTION

Normal cells do not require arginine for growth, since they cansynthesize arginine from citrulline in a two step process catalyzed byargininosuccinate synthase and argininosuccinate lyase. In contrast,melanomas, hepatomas and some sarcomas do not express arginosuccinatesynthase; therefore, they are auxotrophic for arginine. This metabolicdifference may be capitalized upon to develop a safe and effectivetherapy to treat these forms of cancer. Arginine deiminase catalyzes theconversion of arginine to citrulline, and may be used to eliminatearginine. Thus, arginine deiminase may be utilized as a treatment formelanomas, hepatomas and some sarcomas.

Native arginine deiminase may be found in microorganisms and isantigenic and rapidly cleared from circulation in a patient. Theseproblems may be overcome by covalently modifying arginine deiminase withpolyethylene glycol (PEG). Arginine deiminase covalently modified withpolyethylene glycol (with or without a linking group) may be hereinafterreferred to as “ADI-PEG.” When compared to native arginine deiminase,ADI-PEG retains most of its enzymatic activity, is far less antigenic,has a greatly extended circulating half-life, and is much moreefficacious in the treatment of tumors.

“Polyethylene glycol” or “PEG” refers to mixtures of condensationpolymers of ethylene oxide and water, in a branched or straight chain,represented by the general formula H(OCH₂CH₂)_(n)OH, wherein n is atleast 4. “Polyethylene glycol” or “PEG” is used in combination with anumeric suffix to indicate the approximate weight average molecularweight thereof. For example, PEG5,000 refers to polyethylene glycolhaving a total weight average molecular weight of about 5,000; PEG12,000refers to polyethylene glycol having a total weight average molecularweight of about 12,000; and PEG20,000 refers to polyethylene glycolhaving a total weight average molecular weight of about 20,000.

“Melanoma” may be a malignant or benign tumor arising from themelanocytic system of the skin and other organs, including the oralcavity, esophagus, anal canal, vagina, leptomeninges, and/or theconjunctivae or eye. The term “melanoma” includes, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma, subungual melanoma and superficial spreading melanoma.

“Hepatoma” may be a malignant or benign tumor of the liver, including,for example, hepatocellular carcinoma.

“Patient” refers to an animal, preferably a mammal, more preferably ahuman.

“Biocompatible” refers to materials or compounds which are generally notinjurious to biological functions and which will not result in anydegree of unacceptable toxicity, including allergenic and diseasestates.

Throughout the present disclosure, the following abbreviations may beused: PEG, polyethylene glycol; ADI, arginine deiminase; SS,succinimidyl succinate; SSA, succinimidyl succinamide; SPA, succinimidylpropionate; and NHS, N-hydroxy-succinimide.

The present invention is based on the unexpected discovery that ADImodified with polyethylene glycol provides excellent results in treatingcertain types of cancer and inhibiting the metastasis of cancer. ADI maybe covalently bonded to polyethylene glycol with or without a linkinggroup, although a preferred embodiment utilizes a linking group.

In the present invention, the arginine deiminase gene may be derived,cloned or produced from any source, including, for example,microorganisms, recombinant biotechnology or any combination thereof.For example, arginine deiminase may be cloned from microorganisms of thegenera Mycoplasma, Clostridium, Bacillus, Borrelia, Enterococcus,Streptococcus, Lactobacillus, Qiardia. It is preferred that argininedeiminase is cloned from Mycoplasma pneumoniae, Mycoplasma hominus,Mycoplasma arginini, Steptococcus pyogenes, Steptococcus pneumoniae,Borrelia burgdorferi, Borrelia afzelii, Qiardia intestinalis,Clostridium perfringens, Bacillus licheniformis, Enterococcus faecalis,Lactobacillus sake, or any combination thereof. In particular, thearginine deiminase used in the present invention may have one or more ofthe amino acid sequences depicted in FIGS. 1 and 10-13.

In certain embodiments of the present invention, it is preferred thatarginine deiminase is cloned from microorganisms of the genusMycoplasma. More preferably, the arginine deiminase is cloned fromMycoplasma arginini, Mycoplasma hominus, Mycoplasma arthritides, or anycombination thereof. In particular, the arginine deiminase used in thepresent invention may have one or more of the amino acid sequencesdepicted in FIG. 1.

In one embodiment of the present invention, the polyethylene glycol(PEG) has a total weight average molecular weight of about 1,000 toabout 50,000; more preferably from about 3,000 to about 40,000, morepreferably from about 5,000 to about 30,000; more preferably from about8,000 to about 30,000; more preferably from about 11,000 to about30,000; even more preferably from about 12,000 to about 28,000; stillmore preferably from about 16,000 to about 24,000; even more preferablyfrom about 18,000 to about 22,000; even more preferably from about19,000 to about 21,000, and most preferably about 20,000. Generally,polyethylene glycol with a molecular weight of 30,000 or moreis-difficult to dissolve, and yields of the formulated product aregreatly reduced. The polyethylene glycol may be a branched or straightchain, preferably a straight chain. Generally, increasing the molecularweight of the polyethylene glycol decreases the immunogenicity of theADI. The polyethylene glycol having a molecular weight described in thisembodiment may be used in conjunction with ADI, and, optionally, abiocompatible linking group, to treat cancer, including, for example,melanomas, hepatomas and sarcomas, preferably melanomas.

In another embodiment of the present invention, the polyethylene glycolhas a total weight average molecular weight of about 1,000 to about50,000; preferably about 3,000 to about 30,000; more preferably fromabout 3,000 to about 20,000; more preferably from about 4,000 to about12,000; still more preferably from about 4,000 to about 10,000; evenmore preferably from about 4,000 to about 8,000; still more preferablyfrom about 4,000 to about 6,000; with about 5,000 being most preferred.The polyethylene glycol may be a branched or straight chain, preferablya straight chain. The polyethylene glycol having a molecular weightdescribed in this embodiment may be used in conjunction with ADI, andoptionally, a biocompatible linking group, to treat cancer, including,for example, melanomas, hepatomas and sarcomas, preferably hepatomas.

The linking group used to covalently attach ADI to PEG may be anybiocompatible linking group. As discussed above, “biocompatible”indicates that the compound or group is non-toxic and may be utilized invitro or in vivo without causing injury, sickness, disease or death. PEGcan be bonded to the linking group, for example, via an ether bond, anester bond, a thiol bond or an amide bond. Suitable biocompatiblelinking groups include, for example, an ester group, an amide group, animide group, a carbamate group, a carboxyl group, a hydroxyl group, acarbohydrate, a succinimide group (including, for example, succinimidylsuccinate (SS), succinimidyl propionate (SPA), succinimidylcarboxymethylate (SCM), succinimidyl succinamide (SSA) or N-hydroxysuccinimide (NHS)), an epoxide group, an oxycarbonylimidazole group(including, for example, carbonyldimidazole (CDI)), a nitro phenyl group(including, for example, nitrophenyl carbonate (NPC) or trichlorophenylcarbonate (TPC)), a trysylate group, an aldehyde group, an isocyanategroup, a vinylsulfone group, a tyrosine group, a cysteine group, ahistidine group or a primary amine. Preferably, the biocompatiblelinking group is an ester group and/or a succinimide group. Morepreferably, the linking group is SS, SPA, SCM, SSA or NHS; with SS, SPAor NHS being more preferred, and with SS or SPA being most preferred.

Alternatively, ADI may be coupled directly to PEG (i.e., without alinking group) through an amino group, a sulfhydral group, a hydroxylgroup or a carboxyl group.

ADI may be covalently bonded to PEG, via a biocompatible linking group,using methods known in the art, as described, for example, by Park etal, Anticancer Res., 1:373-376 (1981); and Zaplipsky and Lee,Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications,J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992), the disclosuresof which are hereby incorporated by reference herein in their entirety.

The attachment of PEG to ADI increases the circulating half-life of ADI.Generally, PEG is attached to a primary amine of ADI. Selection of theattachment site of polyethylene glycol on the arginine deiminase isdetermined by the role of each of the sites within the active domain ofthe protein, as would be known to the skilled artisan. PEG may beattached to the primary amines of arginine deiminase without substantialloss of enzymatic activity. For example, ADI cloned from Mycoplasmaarginini, Mycoplasma arthritides and Mycoplasma hominus has about 17lysines that may be modified by this procedure. In other words, the 17lysines are all possible points at which ADI can be attached to PEG viaa biocompatible linking group, such as SS, SPA, SCM, SSA and/or NHS. PEGmay also be attached to other sites on ADI, as would be apparent to oneskilled in the art in view of the present disclosure.

From 1 to about 30 PEG molecules may be covalently bonded to ADI.Preferably, ADI is modified with about 7 to about 15 PEG molecules, morepreferably from about 9 to about 12 PEG molecules. In other words, about30% to about 70% of the primary amino groups in arginine deiminase aremodified with PEG, preferably about 40% to about 60%, more preferablyabout 45% to about 55%, and most preferably about 50% of the primaryamino groups in arginine deiminase are modified with PEG. When PEG iscovalently bonded to the end terminus of ADI, preferably only 1 PEGmolecule is utilized. Increasing the number of PEG units on ADIincreases the circulating half life of the enzyme. However, increasingthe number of PEG units on ADI decreases the specific activity of theenzyme. Thus, a balance needs to be achieved between the two, as wouldbe apparent to one skilled in the art in view of the present disclosure.

In the present invention, a common feature of the most preferredbiocompatible linking groups is that they attach to a primary amine ofarginine deiminase via a maleimide group. Once coupled with argininedeiminase, SS-PEG has an ester linkage next to the PEG, which may renderthis site sensitive to serum esterase, which may release PEG from ADI inthe body. SPA-PEG and PEG2-NHS do not have an ester linkage, so they arenot sensitive to serum esterase.

In the present invention, the particular linking groups do not appear toinfluence the circulating half-life of PEG-ADI or its specific enzymeactivity. However, it is critical to use a biocompatible linking groupin the present invention. PEG which is attached to the protein may beeither a single chain, as with SS-PEG, SPA-PEG and SC-PEG, or a branchedchain of PEG may be used, as with PEG2-NHS. The structural formulas ofthe preferred linking groups in the present invention are set forthbelow.

A therapeutically effective amount of one of the compounds of thepresent invention is an amount that is effective to inhibit tumorgrowth. Generally, treatment is initiated with small dosages which canbe increased by small increments until the optimum effect under thecircumstances is achieved. Generally, a therapeutic dosage of compoundsof the present invention may be from about 1 to about 200 mg/kg twice aweek to about once every two weeks. For example, the dosage may be about1 mg/kg once a week as a 2 ml intravenous injection to about 20 mg/kgonce every 3 days. The optimum dosage with ADI-SS-PEG5,000 may be abouttwice a week, while the optimum dosage with ADI-SS-PEG20,000 may be fromabout once a week to about once every two weeks. PEG-ADI may be mixedwith a phosphate buffered saline solution, or any other appropriatesolution known to those skilled in the art, prior to injection. ThePEG-ADI formulation may be administered as a solid (lyophilate) or as aliquid formulation, as desired.

The methods of the present invention can involve either in vitro or invivo applications. In the case of in vitro applications, including cellculture applications, the compounds described herein can be added to thecells in cultures and then incubated. The compounds of the presentinvention may also be used to facilitate the production of monoclonaland/or polyclonal antibodies, using antibody production techniques wellknown in the art. The monoclonal and/or polyclonal antibodies can thenbe used in a wide variety of diagnostic applications, as would beapparent to one skilled in the art.

The in vivo means of administration of the compounds of the presentinvention will vary depending upon the intended application. As oneskilled in the art will recognize, administration of the PEG-ADIcomposition of the present invention can be carried out, for example,orally, intranasally, intraperitoneally, parenterally, intravenously,intralymphatically, intratumorly, intramuscularly, interstitially,intra-arterially, subcutaneously, intraocularly, intrasynovial,transepithelial, and transdermally.

EXAMPLES

The invention is further demonstrated in the following examples, whichare for purposes of illustration, and are not intended to limit thescope of the present invention.

Example 1 Production of Recombinant ADI

Cultures of Mycoplasma arginini (ATCC 23243), Mycoplasma hominus (ATCC23114) and Mycoplasma arthritides (ATCC 23192) were obtained from theAmerican Type Culture Collection, Rockville, Md.

Arginine deiminase was cloned from Mycoplasma arginini, Mycoplasmahominus and Mycoplasma arthritides and expressed in E. coli aspreviously described by S. Misawa et al, J. Biotechnology, 36:145-155(1994), the disclosure of which is hereby incorporated herein byreference in its entirety. The amino acid sequences of argininedeiminase from each of the above species is set forth in FIG. 1. The topamino acid sequence, identified as ADIPROT, is from Mycoplasma arginini;the middle amino acid sequence, identified as ARTADIPRO, is fromMycoplasma arthritides; and the bottom amino acid sequence, identifiedas HOMADIPRO, is from Mycoplasma hominus. Each of the amino acidsequences are more than 96% conserved. Characterization, by methodsknown to those skilled in the art, of each of the proteins with respectto specific enzyme activity, K_(m), V_(max) and pH optima revealed thatthey were biochemically indistinguishable from each other. The pH optimawas determined using a citrate buffer (pH 5-6.5), a phosphate buffer (pH6.5-7.5) and a borate buffer (pH 7.5-8.5). The K_(m) and V_(max) weredetermined by incubating the enzyme with various concentrations ofarginine and quantifying citrulline production. The K_(m) for thevarious enzymes was about 0.02 to 0.06 μM and the V_(max) was about15-20 μmol/min/mg, the values of which are within standard error of eachother.

The arginine deiminase genes were amplified by polymerase chain reactionusing the following primer pair derived from the published sequence ofM. arginini, as described, for example, by T. Ohno et al, Infect.Immun., 58:3788-3795 (1990), the disclosure of which is herebyincorporated by reference herein in its entirety: SEQ ID NO: 45′-GGGATCCATGTCTGTATTTGACAGT-3′, SEQ ID NO: 55′-TGAAAGCTTTTACTACCACTTAACATCTTTACG-3′,The polymerase chain reaction products were cloned as a Bam HI-Hind IIIfragment into expression plasmid pQE16. DNA sequence analysis indicatedthat the fragment derived from M. arginini by PCR had the same sequencefor the arginine deiminase gene as described by Ohno et al, Infect.Immun., supra. The five TGA codons in the ADI gene which encodetryptophan in Mycoplasma were changed to TGG codons byoligonucleotide-directed mutagenesis prior to gene expression in E.coli, as taught, for example, by J. R. Sayers et al, Biotechniques,13:592-596 (1992). Recombinant ADI was expressed in inclusion bodies atlevels of 10% of total cell protein.

The proteins from each of the above three species of Mycoplasma haveapproximately 95% homology and are readily purified by columnchromatography. Approximately 200 mg of pure protein may be isolatedfrom 1 liter of fermentation broth. Recombinant ADI is stable for about2 weeks at 37° C. and for at least 8 months when stored at 4° C. Asdetermined by methods known to those skilled in the art, the proteinshad a high affinity for arginine (0.04 μM), and a physiological pHoptima of about 7.2 to about 7.4.

Example 2 Renaturation and Purification of Recombinant ADI

ADI protein was renatured, with minor modifications, as described byMisawa et al, J. Biotechnology, 36:145-155 (1994), the disclosure ofwhich is hereby incorporated herein by reference in its entirety. 100 gof cell paste was resuspended in 800 ml of 10 mM K₂PO₄ pH 7.0, 1 mM EDTA(buffer 1) and the cells were disrupted by two passes in aMicrofluidizer (Microfluidics Corporation, Newton, Mass.). Triton X-100was added to achieve a final concentration of 4% (v/v). The homogenatewas stirred for 30 min at 4° C., then centrifuged for 30 min at 13,000g. The pellet was collected and resuspended in one liter of buffer 1containing 0.5% Triton X-100. The solution was diafiltered against 5volumes of denaturation buffer (50 mM Tris HCl, pH 8.5, 10 mM DTT) usinghollow-fiber cartridges with 100 kD retention rating (Microgon Inc.,Laguna Hills, Calif.). Guanidine HCl was added to achieve a finalconcentration of 6 M and the solution was stirred for 15 min at 4° C.The solution was diluted 100-fold into refolding buffer 1, 10 mm K₂PO₄,pH 7.0 and stirred for 48 hours at 15° C., particulates were removed bycentrifugation at 15,000×g.

The resulting supernatant was concentrated on a Q Sepharose Fast Flow(Pharmacia Inc., Piscataway, N.J.) column preequilabrated in refoldingbuffer. ADI was eluted using refolding buffer containing 0.2 M NaCl. Thepurification procedure yielded ADI protein, which was >95% pure asestimated by SDS-PAGE analysis. 8 g of pure renatured ADI protein wasproduced from 1 kg of cell paste which corresponds to 200 mg purifiedADI per liter of fermentation.

ADI activity was determined by micro-modification of the methoddescribed by Oginsky et al, Meth. Enzymol., (1957)3:639-642. 10 μlsamples in 0.1 m Na₂PO₄, pH 7.0 (BUN assay buffer) were placed in a 96well microliter plate, 40 μl of 0.5 mM arginine in BUN assay buffer wasadded, and the plate was covered and incubated at 37° C. for 15 minutes.20 μl of complete BUN reagent (Sigma Diagnostics) was added and theplate was incubated for 10 minutes at 100° C. The plate was then cooledto 22° C. and analyzed at 490 nm by a microliter plate reader (MolecularDevices, Inc). 1.0 IU is the amount of enzyme which converts 1 μmole ofL-arginine to L-citrulline per minute. Protein concentrations weredetermined using Pierce Coomassie Blue Protein Assay Reagent (PierceCo., Rockford, Ill.) with bovine serum albumin as a standard.

The enzyme activity of the purified ADI preparations was 17-25 IU/mg.

Example 3 Attachment of PEG to ADI

PEG was covalently bonded to ADI in a 100 mM phosphate buffer, pH 7.4.Briefly, ADI in phosphate buffer was mixed with a 100 molar excess ofPEG. The reaction was stirred at room temperature for 1 hour, then themixture was extensively dialized to remove unincorporated PEG.

A first experiment was performed where the effect of the linking groupused in the PEG-ADI compositions was evaluated. PEG and ADI werecovalently bonded via four different linking groups: an ester group ormaleimide group, including SS, SSA, SPA and SSPA, where the PEG had atotal weight average molecular weight of 5,000, 10,000, 12,000, 20,000,30,000 and 40,000; an epoxy group, PEG-epoxy, where the PEG had a totalweight average molecular weight of 5,000; and a branched PEG group,PEG2-NHS, where the PEG had a total weight average molecular weight of10,000, 20,000 and 40,000.

5.0 IU of the resulting compositions were injected into mice (5 mice ineach group). To determine the serum levels of arginine, the mice werebled from the retro orbital plexus (100 ul). Immediately followingcollection an equal volume of 50% (w/v) of trichloroacetic acid wasadded. The precipitate was removed by centrifugation (13,000×g for 30minutes) and the supernatant removed and stored frozen at −70° C. Thesamples were then analyzed using an automated amino acid analyzer andreagents from Beckman Instruments using protocols supplied by themanufacturer. The limits of sensitivity for arginine by this method wasapproximately 2-6 μM and the reproducibility of measurements withinabout 8%. The amount of serum arginine was determined by amino acidanalysis. As can be seen from the results in FIG. 3, the linking groupcovalently bonding the PEG and ADI did not have an appreciable effect onthe ability of ADI to reduce serum arginine in vivo. In other words, thelinking group may not be critical to the results of the experiment,except that a non-toxic linking group must be used for in vivoapplications.

A second experiment was performed wherein the effect of the linkinggroup and molecular weight of PEG on serum citrulline levels in vivo wasevaluated. Mice (5 in each group) were given various compositions of ADIand PEG-ADI in an amount of 5.0 IU. To determine the serum levels ofcitrulline, the mice were bled from the retro orbital plexus (100 ul).Immediately following collection an equal volume of 50% (w/v) oftrichloroacetic acid was added. The precipitate was removed bycentrifugation (13,000×g for 30 minutes) and the supernatant removed andstored frozen at −70° C. The samples were then analyzed using anautomated amino acid analyzer and reagents from Beckman Instrumentsusing protocols supplied by the manufacturer. The limits of sensitivityfor citrulline by this method was approximately 2-6 μM and thereproducibility of measurements within about 8%. The amount ofcitrulline was determined, and the area under the curve approximated andexpressed as μmol days.

In FIG. 4, the open circles indicate the amount of citrulline producedby native ADI, the filled circles are ADI-SC-PEG, the open squares areADI-SS-PEG, the open triangles are ADI-SPA-PEG, and the filled trianglesare branched chain PEG-NHS-PEG₂. The results in FIG. 4 demonstrate thatthe molecular weight of the PEG determines the effectiveness of thePEG-ADI composition. The effectiveness of the PEG-ADI compositions isnot necessarily based on the method or means of attachment of the PEG toADI, except that a biocompatible linking group must be used for in vivoapplications.

The results in FIG. 4 also demonstrate that the optimal molecular weightof PEG is 20,000. Although PEG30,000 appears to be superior to PEG20,000in terms of its pharmacodynamics, PEG30,000 is less soluble, which makesit more difficult to work with. The yields, which were based on therecovery of enzyme activity, were about 90% for PEG5,000 and PEG12,000;about 85% for PEG20,000 and about 40% for PEG30,000. Therefore,PEG20,000 is the best compromise between yield and circulating halflife, as determined by citrulline production.

In a third experiment, the dose response of serum arginine depletion andthe production of citrulline with ADI-SS-PEG5,000 and ADI-SS-PEG20,000was determined. Mice (5 in each group) were given a single injection of0.05 IU, 0.5 IU or 5.0 IU of either ADI-SS-PEG5,000 or ADI-SS-PEG20,000.At indicated times, serum was collected, as described above, and anamino acid analysis was performed to quantify serum arginine (FIGS. 5Aand 5C) and serum citrulline (FIGS. 5B and 5D). Both formulationsinduced a dose dependent decrease in serum arginine and an increase inserum citrulline. However, the effects induced by ADI-SS-PEG20,000 weremore pronounced and of longer duration than the effects induced byADI-SS-PEG5,000.

Example 4 Selectivity of ADI Mediated Cytotoxicity

The selectivity of arginine deiminase mediated cytotoxicity wasdemonstrated using a number of human tumors. Specifically, human tumorswere tested in vitro for sensitivity to ADI-SS-PEG5,000 (50 ng/ml).Viability of cultures was determined after 7 days. For a culture to bedefined as “inhibited,” greater than 95% of the cells must take upTrypan blue dye. A host of normal cells were also tested, includingendothelial cells, smooth muscle cells, epithelial cells andfibroblasts, and none were inhibited by ADI-SS-PEG5,000. Althougharginine deiminase has no appreciable toxicity towards normal, and mosttumor cells, ADI-SS-PEG5,000 greatly inhibited all human melanomas andhepatomas that were commercially available from the ATCC, MSKCC andEurope. TABLE 1 Specificity of Arginine Deiminase Cytotoxicity TumorType Number of Tumors Tested Tumors inhibited (%) Brain 16 0 Colon 34 0Bladder 3 0 Breast 12 0 Kidney 5 0 Sarcoma 11 64 Hepatoma 17 100Melanoma 37 100

In a parallel set of experiments, mRNA was isolated from the tumors.Northern blot analyses, using the human argininosuccinate synthase cDNAprobe, indicated complete concordance between the sensitivity toarginine deiminase treatment and an inability to expressargininosuccinate synthase. This data suggests that ADI toxicity resultsfrom an inability to induce argininosuccinate synthase. Therefore, thesecells cannot synthesize arginine from citrulline, and are unable tosynthesize the proteins necessary for growth.

Example 5 Circulating Half-Life

Balb C mice (5 in each group) were injected intravenously with a single5.0 IU dose of either native arginine deiminase or various formulationsof arginine deiminase modified with polyethylene glycol, as indicated inFIGS. 2A and 2B. To determine the serum levels of arginine andcitrulline, the mice were bled from the retro orbital plexus (100 μl).Immediately following collection an equal volume of 50% (w/v) oftrichloro-acetic acid was added. The precipitate was removed bycentrifugation (13,000×g for 30 minutes) and the supernatant removed andstored frozen at −70° C. The samples were then analyzed using anautomated amino acid analyzer and reagents from Beckman Instrumentsusing protocols supplied by the manufacturer. The limits of sensitivityfor arginine by this method was approximately 6 μM and thereproducibility of measurements within about 8%.

A dose dependent decrease in serum arginine levels, as shown by thesolid circles in FIG. 2A, and a rise in serum citrulline, as shown bythe open triangles in FIG. 2B, were detected from the single doseadministration of native ADI (filled circles) or ADI-SS-PEG (opentriangles). However, the decrease in serum arginine and rise in serumcitrulline was short lived, and soon returned to normal. The half lifeof arginine depletion is summarized in the Table below. TABLE 2Half-Life of Serum Arginine Depletion Compound Half-Life in Days NativeADI 1 ADI-SS-PEG5,000 5 ADI-SS-PEG12,000 15 ADI-SS-PEG20,000 20ADI-SS-PEG30,000 22

This experiment demonstrates that normal cells and tissues are able toconvert the citrulline back into arginine intracellularly whilemelanomas and hepatomas cannot because they lack argininosuccinatesynthetase.

Example 6 Antigenicity of PEG Modified ADI

To determine the antigenicity of native ADI, ADI-SS-PEG5,000, andADI-SS-PEG20,000, the procedures described in, for example, Park,Anticancer Res., supra, and Kamisaki, J. Pharmacol. Exp. Ther., supra,were followed. Briefly, Balb C mice (5 in each group) were intravenouslyinjected weekly for 12 weeks with approximately 0.5 IU (100 μg ofprotein) of native ADI, ADI-SS-PEG5,000 or ADI-SS-PEG20,000. The animalswere bled (0.05 ml) from the retro orbital plexus at the beginning ofthe experiment and at weeks 4, 8 and 12. The serum was isolated andstored at −70° C. The titers of anti-ADI IgG were determined by ELISA.50 μg of ADI was added to each well of a 96 well micro-titer plate andwas incubated at room temperature for 4 hours. The plates were rinsedwith PBS and then coated with bovine serum albumin (1 mg/ml) to blocknonspecific protein binding sites, and stored over night at 4° C. Thenext day serum from the mice was diluted and added to the wells. After 1hour the plates were rinsed with PBS and rabbit anti-mouse IgG coupledto peroxidase was added to the wells. The plates were incubated for 30min and then the resulting UV absorbance was measured using amicro-titer plate reader. The titer was defined as the highest dilutionof the serum which resulted in a two-fold increase from backgroundabsorbance (approximately 0.50 OD).

The results are shown in FIG. 6. The open circles represent the dataobtained from animals injected with native ADI, which was veryantigenic. The filled circles represent the data obtained from theanimals injected with ADI-SS-PEG5,000, while the open trianglesrepresent the data obtained from the animals injected withADI-SS-PEG20,000. As can be seen from FIG. 6, ADI-SS-PEG5,000 andADI-SS-PEG20,000 are significantly less antigenic than native ADI. Forexample, as few as 4 injections of native ADI resulted in a titer ofabout 10⁶, while 4 injections of any of the PEG-ADI formulations failedto produce any measurable antibody. However, after 8 injections, theADI-PEG5,000 had a titer of about 10², while ADI-PEG20,000 did notinduce this much of an immune response until after 12 injections. Theresults demonstrate that attaching PEG to ADI blunts the immune responseto the protein.

Example 7 Tumor Inhibition of Human Melanomas

The effect of PEG-ADI on the growth of human melanoma (SK-Mel 28) innude mice was determined. Nude mice (5 in each group) were injected with10⁶ SK-mel 2 human melanoma cells which were allowed to grow until thetumors reached a diameter of about 3-5 mm. The mice were left untreated(open circles) or were treated once a week for 8 weeks with 5.0 IU ofADI-SS-PEG5,000 (filled triangles), ADI-SS-PEG12,000 (open triangles) orADI-SS-PEG20,000 (filled circles). The tumor size was measured weekly,and the mean diameter of the tumors is presented in FIG. 7.

FIG. 8 shows the effectiveness of ADI-SS-PEG20,000 on three humanmelanomas (SK-mel 2, SK-mel 28, M24-met) grown in vivo in nude mice.Nude mice (5 in each group) were injected with 10⁶ SK-mel 2, SK-mel 28or M24-met human melanoma cells. The tumors were allowed to grow untilthey were approximately 3-5 mm in diameter. Thereafter, the animals wereinjected once a week with 5.0 IU of ADI-SS-PEG20,000. The results areshown in FIG. 8, and show that PEG-ADI inhibited tumor growth and thateventually the tumors began to regress and disappear. Because the tumorsdid not have argininosuccinate synthatase, they were unable tosynthesize proteins (because ADI eliminated arginine and the tumorscould not make it) so that the cells “starved to death.”

Since M24-met human melanoma is highly metastatic, the animals injectedwith M24-met human melanoma cells were sacrificed after 4 weeks oftreatment and the number of metastases in the lungs of the animals wasdetermined. The control animals had an average of 32 metastases, whilethe animals treated with ADI-SS-PEG20,000 did not have any metastases.The results appear to indicate that ADI-SS-PEG20,000 not only inhibitedthe growth of the primary melanoma tumor, but also inhibited theformation of metastases.

It is of interest to note that in over 200 animals tested, the averagenumber of metastases in the control group was 49±18, while only a singlemetastasis was observed in 1 treated animal.

Example 8 Tumor Inhibition of Human Hepatomas

The ability of PEG-ADI to inhibit the growth of a human hepatoma in vivowas tested. Nude mice (5 in each group) were injected with 10⁶ humanhepatoma SK-Hep1 cells. The tumors were allowed to grow for two weeksand then the animals were treated once a week with 5.0 IU ofSS-PEG5,000-ADI (solid circles), SS-PEG12,000-ADI (solid triangles) orSS-PEG20,000-ADI (open triangles). The results are set forth in FIG. 9.The untreated animals (open circles) all died within 3 weeks. Incontrast, animals treated with ADI had a far longer life expectancy, ascan be seen from FIG. 9. All the surviving mice were euthanized after 6months, and necropsy indicated that they were free of tumors.

Surprisingly, PEG5,000-ADI is most effective in inhibiting hepatomagrowth in vivo. The exact mechanism by which this occurs is unknown.Without being bound to any theory of the invention works, it appearsthat proteins formulated with SS-PEG5,000-ADI become sequestered in theliver. Larger molecular weights of PEG do not, which may be due to theuniqueness of the hepatic endothelium and the spaces (fenestrae) beingof such a size that larger molecular weights of PEG-ADI conjugates areexcluded.

Example 9 Application to Humans

PEG5,000-ADI and PEG20,000-ADI were incubated ex vivo with normal humanserum and the effects on arginine concentration was determined by aminoacid analysis, where the enzyme was found to be fully active and capableof degrading all the detectable arginine with the same kinetics as inthe experiments involving mice. The reaction was conducted at a volumeof 0.1 ml in a time of 1 hour at 37° C. Additionally, the levels ofarginine and citrulline in human serum are identical with that found inmice. PEG-proteins circulate longer in humans than they do in mice. Forexample, the circulating half life of PEG conjugated adenosinedeiminase, asparaginase, glucocerbrocidase, uricase, hemoglobulin andsuperoxide dismutase all have a circulating half life that is 5 to 10times longer than the same formulations in mice. What this has meant inthe past is that the human dose is most often 1/5 to 1/10 of that usedin mice. Accordingly, PEG-ADI should circulate even longer in humansthan it does in mice.

Each of the patents, patent applications and publications describedherein are hereby incorporated by reference herein in their entirety.

Various modifications of the invention, in addition to those describedherein, will be apparent to one skilled in the art in view of theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

1-22. (canceled)
 23. A method of enhancing the circulating half life ofarginine deiminase comprising modifying said arginine deiminase bycovalently bonding said arginine deiminase via a linking group topolyethylene glycol, wherein the arginine deiminase is derived from amicroorganism of the genus selected from the group consisting of:Steptococcus, Borrelia, Qiardia, Clostridium, Enterococcus,Lactobacillus, and Bacillus; wherein the polyethylene glycol has a totalweight average molecular weight of from about 1,000 to about 40,000, andwherein the linking group is selected from the group consisting of asuccinimide group, an amide group, an imide group, a carbarmate group,an ester group, an epoxy group, a carboxyl group, a hydroxyl group, acarbohydrate, a tyrosine group, a cysteine group, a histidine group andcombinations thereof.
 24. A method of enhancing the tumoricidal activityof arginine deiminase comprising modifying said arginine deiminase bycovalently bonding said arginine deiminase via a linking group topolyethylene glycol, wherein the arginine deiminase is derived from amicroorganism of the genus selected from the group consisting of:Steptococcus, Borrelia, Qiardia, Clostridium, Enterococcus,Lactobacillus, and Bacillus; wherein the polyethylene glycol has a totalweight average molecular weight of from about 1,000 to about 40,000, andwherein the linking group is selected from the group consisting of asuccinimide group, an amide group, an imide group, a carbamate group, anester group, an epoxy group, a carboxyl group, a hydroxyl group, acarbohydrate, a tyrosine group, a cysteine group, a histidine group andcombinations thereof.
 25. A method of treating a tumor in a patientcomprising administering to said patient a compound comprising argininedeiminase covalently bonded via a linking group to polyethylene glycol,wherein the arginine deiminase is derived from a microorganism of thegenus selected from the group consisting of: Steptococcus, Borrelia,Qiardia, Clostridium, Enterococcus, Lactobacillus, and Bacillus; whereinthe polyethylene glycol has a total weight average molecular weight offrom about 1,000 to about 40,000, and wherein the linking group isselected from the group consisting of a succinimide group, an amidegroup, an imide group, a carbamate group, an ester group, an epoxygroup, a carboxyl group, a hydroxyl group, a carbohydrate, a tyrosinegroup, a cysteine group, a histidine group and combinations thereof. 26.The method of claim 25, wherein said tumor is a melanoma.
 27. The methodof claim 26, wherein said polyethylene glycol has a total weight averagemolecular weight of about 20,000.
 28. The method of claim 26, whereinsaid linking group is a succinimide group.
 29. The method of claim 28,wherein said succinimide group is succinimidyl succinate, succinimidylpropionate, succinimidyl carboxymethylate, succinimidyl succinamide,N-hydroxy succinimide or combinations thereof.
 30. The method of claim25, wherein said tumor is a hepatoma.
 31. The method of claim 30,wherein said polyethylene glycol has a total weight average molecularweight of about 5,000.
 32. The method of claim 30, wherein said linkinggroup is a succinimide group.
 33. The method of claim 32, wherein saidsuccinimide group is succinimidyl succinate, succinimidyl propionate,succinimidyl carboxymethylate, succinimidyl succinamide, N-hydroxysuccinimide or combinations thereof.
 34. The method of claim 25, whereinsaid tumor is a sarcoma.
 35. A method of treating and inhibitingmetastases in a patient comprising administering to said patient acompound comprising arginine deiminase covalently bonded via a linkinggroup to polyethylene glycol, wherein the arginine deiminase is derivedfrom a microorganism of the genus selected from the group consisting of:Steptococcus, Borrelia, Qiardia, Clostridium, Enterococcus,Lactobacillus, and Bacillus; wherein the polyethylene glycol has a totalweight average molecular weight of from about 1,000 to about 40,000, andwherein the linking group is selected from the group consisting of asuccinimide group, an amide group, an imide group, a carbamate group, anester group, an epoxy group, a carboxyl group, a hydroxyl group, acarbohydrate, a tyrosine group, a cysteine group, a histidine group andcombinations thereof.